Purification of recombinant proteins commonly begins with a clarification step in which cells and debris are removed so that the remaining supernatant can be processed by methods that would be hampered or rendered ineffective by the presence of cells and debris. Their removal commonly involves physical methods such as centrifugation and microfiltration. It sometimes involves the use of membrane or depth filters with anion exchange capabilities, or the addition of anion exchange polymers or particles directly to the antibody-containing harvest (Gagnon, P., Purification Tools for Monoclonal Antibodies, Validated Biosystems, Tucson, 1996; Kuczewski, M, et al, Biopharm Int. 23 (3) (2010) 20-25; Kuczewski, M., et al, Biotechnol. J., 6 (2011) 56-65). Gan et al (J. Chromatography A-191 (2013) 33-40) have recently indicated that targeted removal of chromatin catabolites with soluble and insoluble forms of multivalent organic ions supports especially effective conditioning of cell culture harvests. Physical clarification methods typically achieve no significant chromatin reduction. Adding anion exchange particles to the harvest typically removes about half of the DNA. Some of the methods described by Gan et al (supra) remove 99% of chromatin, and may accurately be referred to as chromatin-directed clarification methods.
Purification of proteins by precipitation with polyethylene glycol (PEG) has been described. It is typically performed as an aqueous phase technique, where PEG is dissolved in an aqueous protein preparation and causes the protein to precipitate from that solution. The size and concentration of PEG are known process variables, as is pH (Gagnon 1996 supra; D. Atha, K., et al, J. Biol. Chem., 256 (1981) 12108-12117; U.S. Patent Application Publication No. 2008/0214795, each of which are incorporated herein by reference. The closer the operating pH to the isoelectric point of the antibody, the lower the concentration of PEG required to achieve precipitation. It has been indicated that the concentration of non-protein-precipitating salts such as sodium chloride (NaCl) has little significant effect on selectivity (Atha et al supra), but the technique has been carried out in the presence of NaCl at concentrations up to about 0.1.7 M (10%, Gervais et al., U.S. Patent Application Publication No. 2010/0204455). It has also been demonstrated that PEG-mediated steric exclusion chromatography achieves higher IgG recovery and lower contaminant levels when PEG is combined with elevated concentrations of NaCl, and further noted that it largely suspends the influence of pH (P. Gagnon et al, J. Chromatogr. A 1324 (2014) 171-180). Combining PEG with the protein-precipitating salt sodium phosphate has been described (Gervais, supra; U.S. Pat. Nos. 4,379,086 and 4,515,776).
Removal of residual PEG after the precipitate is re-solubilized is a major challenge for the technique. Kuczewski et al supra removed it by performing cation exchange chromatography under conditions where IgG bound to the cation exchange column while the PEG flowed through. PEG removal is otherwise complicated by the fact that it occupies the same range of sizes, measured as hydrodynamic radius or diameter, as the proteins it is used to precipitate. This disqualifies the standard methods of size exclusion chromatography, dialysis, and diafiltration for PEG removal. Anion exchange chromatography in flow-through mode, as widely practiced with IgG monoclonal antibodies, is also unsuitable because the PEG flows through with the antibody.